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Abstract

Fusarium pathogens are among the most damaging pathogens of cereals. These pathogens have the ability to attack the roots, seedlings, and flowering heads of barley and wheat plants (Simpson et al., 2004). Resulting in yield loss and head blight disease and also resulting in the contamination of grain with mycotoxins harmful to human and animal health (McMulen et al., 1997; Walter et al., 2010; Agostinelli et al., 2012). The study of Fusarium diseases, including host disease resistance and the effect of exogenous agents (chemicals, biocontrol agents, etc.), requires robust and effective methods for the assessment and quantification of visual disease symptoms. Here we describe the methods commonly used for the assessment and quantification of the severity of Fusarium seedling blight and Fusarium head blight disease.

Pass the conidial
suspension through two layers of sterile cheese cloth and wash three
times with sterile distilled water. Resuspend conidia in 0.2% Tween 20
and determine the concentration using disposable KOVA Glasstic Slides
10. Adjust the concentration to 2 x 106 spores/ml 0.2% Tween 20. Note: For spore counting using KOVA Glasstic slides, determine the
average number of spores per small grid and multiply with 90,000 to get
number spores/ml.

Germinate the barley seeds as described above for the Fusarium seedling blight experiments.

Transfer germinated seed to a 15 cm-diameter pot containing John
Innes compost No 2. Cultivate plants in the glasshouse. Add 2 g of NPK
10-10-20 fertiliser to each pot at growth stages (GS; Zadoks et al.,
1974) 20 and 30.Note: For more information on different Zadoks
growth stages go to http://www.cerealcentral.ca/crop-management_cereal-staging.aspx.

Treat the heads at mid-anthesis (GS 65) by injecting 4 µl of conidial
suspension (5x 104 spores/ml 0.2% Tween 20) or mock 0.2% Tween 20
treatment into each of the four middle spikelets of a head.

Immediately cover treated heads with a polythene bag for 48 h in order to promote Fusarium infection.

Score the FHB disease symptoms (Figure 2) (percentages bleached
spikelets per head) at GS 70, 80 and 90 and calculate the Area Under the
Disease Progress Curve (AUDPC) [as described by Shaner and Finney
(1977)].
Where, Yi is an assessment of a disease progression
(percentages bleached spikelets per head) at the ith observation, ti is
time (day) at the ith observation, and n is the total number of
observations (Shaner and Finney, 1977).

When ripe (GS 91),
harvest the cereal heads, freeze-dry for 48 h and determine the number
and weight of grains in each head. Use this data to calculate the 1,000
grain weight.

The glasshouse FHB disease trials should be
conducted at least twice and each trial should include at least 5
replicate plants (2 heads per plant) per treatment combination arranged
in a randomised layout.

Figure 2. Symptoms of Fusarium head blight
in barley (cultivar Akashinriki). Four middle spikelets were injected
with 4 µl of A conidia suspension of Fusarium culmorum (strain FCF 200)
in 0.2% Tween 20, or B 0.2% Tween 20. Disease symptoms (percentages of
bleached spikelets per head) and AUDPC were scored at GS 80. White arrow
indicates the point of inoculation.

Recipes

Mung bean broth
Boil 20 g/L mung bean in distilled water for 20 min
Pass the broth through two layers of cheese cloths and make up the volume up to 1 L
Autoclave for 15-20 min at 121 °C

Acknowledgments

This work was supported by the Science Foundation Ireland research fund (IN10/IN.1/B3028) and Department of Agriculture Research Stimulus Grant RSF 07 513.

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